EP2894474A1 - Procédé et trousse pour la détection de biomarqueurs sanguins du cancer rénal - Google Patents

Procédé et trousse pour la détection de biomarqueurs sanguins du cancer rénal Download PDF

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EP2894474A1
EP2894474A1 EP12884039.4A EP12884039A EP2894474A1 EP 2894474 A1 EP2894474 A1 EP 2894474A1 EP 12884039 A EP12884039 A EP 12884039A EP 2894474 A1 EP2894474 A1 EP 2894474A1
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kidney cancer
blood
antibody
sample
detecting
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German (de)
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EP2894474B1 (fr
EP2894474A4 (fr
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Dong Su Kim
Mi Hyang Moon
Hyung Jin Na
Kyung Min Kim
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Genomine Inc
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Genomine Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91005—Transferases (2.) transferring one-carbon groups (2.1)
    • G01N2333/91011—Methyltransferases (general) (2.1.1.)
    • G01N2333/91017—Methyltransferases (general) (2.1.1.) with definite EC number (2.1.1.-)

Definitions

  • the present invention relates to a method and a kit for detecting renal cancer blood biomarkers.
  • kidney cancer carcinoma which originates in the renal parenchyma (a specialized tissue, composed of kidney cortex and medulla, where urine-producing cells aggregate).
  • renal cell carcinoma occurs as single tumor in either of the kidneys, but occasionally spreads to both the kidneys.
  • Renal cell carcinoma is not a single entity, but rather a collection of different types of tumours, which are classified into clear cell RCC, papillary RCC, chromophobe RCC, medullary RCC, unclassified RCC, kidney transitional cell carcinoma (TCC), and renal oncocytoma, with clear cell RCC accounting for 66 ⁇ 75 % of kidney cancer cases, papillary RCC for approximately 15 %, and chromophoobe RCC for approximately 5 %.
  • renal cell carcinoma is diagnosed to be in stage I when the tumor is limited to the kidney parenchyma, in stage II upon involvement of perinephric fat, but remaining limited to Gerota's fascia, in stage III upon metastasis to perinephric vessels or lymph nodes, and in stage IV upon infiltration into other perinephric organs or distal metastasis.
  • stage I is subdivided into stage Ia and Ib with a tumor cut off size of 4 cm.
  • the tumor is assigned with stage III/a when it infiltrates perinephric vessels such as the renal veins or inferior vena cava, and stage III/b when it metastasizes into perinephric lymph nodes.
  • Kidney cancer is diagnosed most in elderly people in their sixties to seventies, with the increasing tendency of incidence. Kidney cancer accounts for approximately 2.0 % and 1.2 % of all cancers diagnosed in men and women, ranking second and fifteenth, respectively. No symptoms can be detected until the tumor has grown to the degree of pushing out the organ. Hematuria is the most common symptom of kidney cancer, but is observed only in 60 % of the patients. Thus, there may also be no signs or symptoms in a person with kidney cancer, especially in the early stages of the disease. Under this situation, about 30 % of the diagnosed cases are in a stage of metastasis, with the expression of symptoms, such as dyspnea, cough, headache, etc., according to the metastasized sites.
  • Diagnosis of kidney cancer is, for the most part, conducted using X-ray, CT, ultrasonography, etc., followed by confirmation through biopsy. These methods, however, are limited in discriminating various types of kidney cancer, and require much time and labor.
  • Disclosed in the present invention is a method useful for the early and non-invasive diagnosis of kidney cancer in which the three kidney cancer blood biomarkers NNMT (Nicotinamide N-methyltransferase), LCP1 (L-Plastin) and NM23A (Non-metastatic cells 1 protein) are used in combination to increase the diagnosis of kidney cancer.
  • NNMT Nicotinamide N-methyltransferase
  • LCP1 L-Plastin
  • NM23A Non-metastatic cells 1 protein
  • the present invention addresses a method for detecting a kidney cancer blood biomarker.
  • NNMT, LCP1, and NM23A levels were higher in the blood of the patient group than in the healthy group, and thus can be useful as kidney cancer blood biomarkers. Also, a combination of the three kidney cancer blood biomarkers was found to diagnose kidney cancer accurately and effectively as measured by the ROC (Receiver Operating Characteristic) curve analysis.
  • NNMT which has the highest diagnostic ability among the three blood biomarkers (NNMT is better diagnostic for kidney cancer than LCP1 and NM23A; see the following Example Section) exhibits an accuracy (AUC) of 0.927 with a specificity of 95 % and a sensitivity of 69 % while a combination of the three blood biomarkers increased AUC to 0.9333 with a specificity of 95 % and a sensitivity of 77 % when applied to the stepwise logistic regression analysis, and further increased AUC to 0.948 with a specificity of 95 % and a sensitivity of 89 % when applied to the scoring method explained in the following Example Section.
  • AUC accuracy
  • a combination of the kidney cancer blood biomarkers were subjected to a blind test with 30 healthy blood samples and 29 patient blood samples using the scoring method.
  • the method for detecting a kidney cancer blood biomarker in accordance with the present invention.
  • the method according to the present invention comprises:
  • NNMT nicotinamide-N-methyltransferase
  • NM_006169 nucleotide sequence
  • NP_006160.1 amino acid sequence
  • LCP-1(L-Plastin) is a family of actin-binding proteins. In humans, two ubiquitous plastin isofomrs (L and T) have been identified. The L isoform is expressed only in hemopoietic cell lineages. LCP-1 is composed of a total of 627 amino acids, with a molecular weight of 70.8 KDa and is disclosed in GenBank Accession number: NM_002298.4 for the nucleotide sequence and in GenBank Accession number:NP_002289.1 for the amino acid sequence.
  • NM23A is an isoform of the protein NM23, which is known for its reduced mRNA transcript levels in highly metastatic cells. It is composed of a total of 152 amino acids with a molecular weight of 16.9 KDa. Reference may be made to GenBank Accession number: NM_198175.1 for the nucleotide sequence and to GenBank Accession number:NP_000260.1 for the amino acid sequence.
  • the detecting method of the present invention can produce quantitative analysis data about all of the kidney cancer blood biomarkers NNMT, LCP1 and NM23A that can be usefully utilized for medical experts such as physicians to determine the incidence, progression and/or cure of kidney cancer.
  • the three kidney cancer blood biomarkers can be very effectively used to diagnose clear cell RCC, which accounts for a majority of RCC cases, because of its high sensitivity of 96 % (for comparison, a specificity of 93 % was detected in NMMT, which is the highest diagnostic biomarker for kidney cancer).
  • processed sample of blood refers to a plasma or serum sample.
  • kidney cancer blood biomarker means NNMT, LCP1 or NM23A, which can be utilized to discriminate kidney cancer patients from healthy persons because they are present in higher levels in the blood in kidney cancer patients than in healthy persons.
  • binding molecule specific for the kidney cancer blood biomarker refers to any molecule that binds specifically only to the kidney cancer blood biomarker, but not substantially to other proteins and which allows for the detection of the specific binding.
  • the binding molecule specific for the kidney cancer blood biomarker include antibodies and aptamers, with preference for antibodies.
  • substantially means that a nonspecific complex may be formed, although to a very low degree, between the binding molecule and non-targets. Such nonspecific complexes can be washed off with a wash solution before detection of the specific binding, as will be described later.
  • antibody is intended to encompass all forms of a molecule capable of binding specifically to a renal cell carcinoma diagnostic marker according to the present invention.
  • the antibody includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (which recognize two or more antigens or epitopes; e.g., bispecific antibodies), as well as fragments of an antibody molecule, recombinant antibodies and chemically modified antibodies, which retain an ability to specifically bind to any one of the renal cell carcinoma diagnostic markers of the present invention.
  • antibody fragments include Fab, F(ab') 2 , single chain Fv (scFv; consisting of a variable heavy chain and a variable light chain linked via an appropriate linker), Fv, and Fab/c (having one Fab and a complete Fc).
  • the antibody fragments may be obtained by treating a whole antibody with a proteolytic enzyme, such as papain or pepsin.
  • the immunoglobulin isotypes of the above antibodies are not specifically limited as long as they retain the ability to bind specifically to a kidney cancer blood biomarker according to the present invention, and may be any one of IgG, IgM, IgA, IgE and IgD.
  • the antibody binding specifically to the kidney cancer blood biomarker may be a polyclonal antibody or a monoclonal antibody.
  • the preparation of monoclonal or polyclonal antibodies is known in the art.
  • a polyclonal antibody may be prepared by immunizing an animal, such as birds (e.g., chickens, etc.) or mammals (e.g., rabbits, goats, horse, sheep, rats, etc.), with a kidney cancer blood biomarker according to the present invention, followed by purification from the blood of the immunized animal using a method known in the art, such as ammonium sulfate fractionation, ion-exchange chromatography and affinity chromatography.
  • a monoclonal antibody may be obtained by establishing a hybridoma cell line, which secretes a monoclonal antibody specific to a kidney cancer blood biomarker according to the present invention.
  • a hybridoma cell line may be produced by immunizing an animal with a kidney cancer blood biomarker according to the present invention, extracting antibody-producing cells such as splenocytes, lymph node cells, etc., from the immunized animal, fusing the antibody-producing cells with a myeloma cell line to produce hybridoma cells from the fused cells, and identifying a hybridoma cell line producing a desired monoclonal antibody.
  • the monoclonal antibody is then recovered from the hybridoma cells using a method known in the art.
  • the detecting step of a complex between a kidney cancer marker and an antibody may be achieved by an immunological analysis method known in the art, such as ELISA (enzyme-linked immunosorbent assay), sandwich immunoassay, fluoroimmunoassay, luminescent immunoassay, etc.
  • an immunological analysis method known in the art, such as ELISA (enzyme-linked immunosorbent assay), sandwich immunoassay, fluoroimmunoassay, luminescent immunoassay, etc.
  • an enzyme-conjugated secondary antibody may be used in ELISA.
  • the enzyme available in ELISA include peroxidase (POD), alkaline phosphatase, ⁇ -galactosidase, horseradish peroxidase, urease, catalase, glucose oxidase, lactate dehydrogenase, and amylase.
  • POD peroxidase
  • alkaline phosphatase alkaline phosphatase
  • ⁇ -galactosidase horseradish peroxidase
  • urease catalase
  • glucose oxidase lactate dehydrogenase
  • amylase amylase
  • the fluoroimmunoassay may be performed using an antibody conjugated with a fluorescent substance or a fluorophore, such as fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, substituted rhodamine isothiocyanate, dichlorotriazine isothiocyanate, Alexa, or AlexaFluoro.
  • a fluorescent substance or a fluorophore such as fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, substituted rhodamine isothiocyanate, dichlorotriazine isothiocyanate, Alexa, or AlexaFluoro.
  • the luminescent immunoassay may be carried out with a luciferase system, a luminol-hydrogen peroxide-POD system, a dioxetane compound system, or the like.
  • the method of the present invention may be conducted using sandwich immunoassay.
  • Examples of the support available in the present invention include, but are not limited to, microspheres (resin beads, magnetic beads, etc.), particles (metal fine particles, gold colloids, etc.), and plates (microtiter plates, glass plates, silicon plates, resin plates, electrode plates, membranes, etc.).
  • the support may be made of (i) an inorganic material, such as glass, quartz glass, alumina, sapphire, forsterite, silica, etc., or (ii) an organic material such as polyethylene, polyvinyl acetal, an acrylic resin, polycarbonate, a phenol resin, an urea resin, en epoxy resin, a melamine resin, a silicon resin, polyphenylene oxide, polysulfone, polyethylene glycol, agarose, acrylamide, nitrocellulose, nylon, latex, etc.
  • an inorganic material such as glass, quartz glass, alumina, sapphire, forsterite, silica, etc.
  • an organic material such as polyethylene, polyvinyl acetal, an acrylic resin, polycarbonate, a phenol resin, an urea resin, en epoxy resin, a melamine resin, a silicon resin, polyphenylene oxide, polysulfone, polyethylene glycol, agarose, acrylamide, nitrocellulose, nylon, late
  • Immobilization of the capturing antibody onto the support may be achieved directly by adsorption (e.g., coating) or indirectly through a linker that binds to both the protein and the support.
  • the adsorption by which the capturing antibody is immobilized onto the support may be carried out by diluting the capturing antibody in a 0.06 M carbonate or bicarbonate buffer, pH 9.5, and bringing the dilution into contact with the support at a certain temperature for a predetermined period of time.
  • the time and the temperature necessary for the adsorption are not imparted with particular limitations so long as those allows for sufficient adsorption.
  • adsorption may be performed for 72 hrs.
  • 2 hrs of adsorption may be given at 37°C.
  • the support may be rinsed with distilled water or ethanol and further coated with a blocking agent such as bovine serum albumin (BSA) in a buffer, e.g., PBS.
  • a blocking agent such as bovine serum albumin (BSA) in a buffer, e.g., PBS.
  • the support may be washed with distilled water or a buffer containing no blocking agents.
  • the capturing antibody adsorbed onto the support forms a complex with a kidney cancer blood biomarker contained in the sample.
  • the support be rinsed with a wash buffer such as Tween 20, or distilled water so as to remove an antibody that would be bound non-specifically, or a contaminant.
  • the label may be a chemical such as biotin, a fluorescent such as fluorescein isothianate, tetramethyl rhodamine isothiocyanate, etc., or a radioisotope such as iodine ( 131 I, 125 I, 123 I, 121 I), phosphorus ( 32 P), sulfur ( 35 S), etc.
  • a fluorescent such as fluorescein isothianate, tetramethyl rhodamine isothiocyanate, etc.
  • a radioisotope such as iodine ( 131 I, 125 I, 123 I, 121 I), phosphorus ( 32 P), sulfur ( 35 S), etc.
  • the enzyme may catalyze a reaction responsible for color development, fluorescence, or luminescence, like peroxidase (POD), alkaline phosphatase, ⁇ -galactosidase, horseradish peroxidase, urease, catalase, glucose oxidase, lactate dehydrogenase, amylase, luciferase, etc.
  • POD peroxidase
  • alkaline phosphatase alkaline phosphatase
  • ⁇ -galactosidase horseradish peroxidase
  • urease catalase
  • glucose oxidase lactate dehydrogenase
  • amylase luciferase
  • the label or the enzyme may be covalently bonded to the antibody.
  • detection antibody those that recognize the Fc region of the antibody (capturing antibody). These detection antibodies may be obtained by immunizing an animal, such as a bird, a mammal, etc, with an Fc region, followed by separation and purification from the blood of the animal.
  • the signal measurement may be obtained using a method known in the art.
  • biotin for example, avidin or streptavidin may be conjugated to measure signals.
  • a substrate thereto may be adopted for signal measurement.
  • luciferin may be used for luciferase.
  • the method of the present invention may preferably further comprise washing the support with a wash buffer such as Tween 20 or distilled water to remove non-specifically bound antibodies or contaminants, as mentioned above.
  • a wash buffer such as Tween 20 or distilled water
  • the method of the present invention may be performed using ELISA.
  • the enzyme may catalyze a reaction responsible for color development, fluorescence, or luminescence, as described above.
  • the secondary antibody may be obtained by immunizing a mammal with the primary antibody as an antigen, followed by separation and purification from the blood of the animal.
  • the step of measuring the activity of the enzyme may be accomplished by adding a substrate to the enzyme and analyzing the enzymatic reaction.
  • the molecule binding specifically to the kidney cancer blood biomarker may be an aptamer binding specifically to the kidney cancer blood biomarker.
  • the aptamer may be an oligonucleotide or peptide molecule.
  • antamers reference may be made to documents ( Bock LC et al., Nature 355(6360):5646(1992 ); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine”. J Mol Med. 78(8):42630(2000 ); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24):142727(1998 )).
  • contemplated in accordance with another aspect of the present invention is a kit for detecting a kidney cancer blood biomarker.
  • the kit of the present invention may include (a) respective molecules binding specifically to kidney cancer blood biomarkers NNMT, LCP1, and NM23A, and (b) a manual that teaches the detection of NNMT, LCP1 and NM23A and the use of blood levels of the kidney cancer blood biomarkers in diagnosing kidney cancer.
  • the molecules specific for the kidney cancer blood biomarkers are antibodies or aptamers, the former being preferred.
  • the molecules specific for the kidney cancer blood biomarkers may be in a form immobilized onto a support or as isolates.
  • the kit may further include a support.
  • the support may be a microsphere, a particle, or a plate, with a preference for a microsphere. More preferable is a resin bead.
  • the kit of the present invention may further include a secondary antibody or a detection antibody in a form conjugated with a label or enzyme allowing for signal measurement, or as an isolate.
  • the kit of the present invention may further include the label or enzyme.
  • the kit of the present invention may further include a wash buffer, a buffer for diluting the samples or the antibodies, an enzyme substrate, a reaction stopper, etc.
  • kidney cancer blood biomarkers NNMT, LCP1 and NM23A in diagnosing kidney cancer, as taught in the manual, may be accomplished in various manners. For instance, when respective blood levels of NNMT, LCP1, and NM23A are compared with cut-off values calculated by logistic regression analysis, the subject is diagnosed with kidney cancer if the blood levels are greater than the cut-off values, and classified as a healthy person if the blood levels are the same as or less than the cut-off values. Alternatively, results obtained according to the scoring method described in the following Example Section may be compared with cut-off values. The subject may be diagnosed with kidney cancer if the results are greater than the cut-off values, or as being healthy if otherwise.
  • the cut-off values are criteria for classifying the subject as a patient or a normal person, and may be arbitrarily determined in consideration of diagnostic factors including specificity, sensitivity, etc.
  • the cut-off may be determined as a value at which a specificity of 95 % is obtained as measured by ROC curve analysis or as a value at which a maximum AUC is obtained on an ROC curve (that is, a value at which an area established by two imaginary vertical and horizontal lines passing a point on an ROC curve is maximized).
  • the present invention addresses a method for detecting LCP1 serving as a kidney cancer blood biomarker, the method comprising (a) treating a blood sample from a subject or a processed sample thereof with a binding molecule specific for the kidney cancer blood biomarker LCP1 to form a complex between the kidney cancer blood biomarker LCP1 and the binding molecule, and (b) detecting the complex.
  • the present invention addresses a method for detecting NM23A serving as a kidney cancer blood biomarker, the method comprising (a) treating a blood sample from a subject or a processed sample thereof with a binding molecule specific for the kidney cancer blood biomarker NM23A to form a complex between the kidney cancer blood biomarker NM23A and the binding molecule, and (b) detecting the complex.
  • LCP1 and NM23A have been known as kidney cancer biomarkers from tissue samples (Korean Unexamined Patent Application Publication No. 10-2009-0014979 ), but not from blood until the present invention.
  • kidney cancer blood biomarker LCP1 or NM23A To details of the detecting method of the kidney cancer blood biomarker LCP1 or NM23A, the foregoing description may be applied as it is.
  • the present invention provides a method and a kit for detecting a kidney cancer blood biomarker.
  • the detecting method and kit according to the present invention can produce quantitative analysis data about all of the kidney cancer blood biomarkers NNMT, LCP1, and NM23A that can be usefully utilized by medical experts such as physicians to determine the incidence, progression and/or cure of kidney cancer.
  • the detecting method and kit can be very effectively used for the early diagnosis of kidney cancer and clear cell RCC, which accounts for a majority of RCC cases.
  • kidney cancer patients had an average age of 55.9 years and could be classified, according to types of kidney cancer, into: a clear cell renal cell carcinoma (clear cell RCC) group of 89 patients; papillary RCC of 6 patients; chromophobe RCC of 7 patients; kidney transitional cell carcinoma (TCC) of 8 patients; Ewing sarcoma of 1 patient; and unclassified RCC of 3 patients.
  • clear cell RCC clear cell renal cell carcinoma
  • TCC kidney transitional cell carcinoma
  • kidney cancer The remainders were accounted for by 6 patients with benign tumor of non-renal oncocytoma or with kidney cancer-negative diagnosis, and 9 patients with benign tumors. According to the progression of kidney cancer, the 129 patients could be divided into: 59 in stage Ia/Ib; 13 in stage II; 29 in stage IIIa/IIIb; and 2 in stage IV; the remainder was not classified.
  • Normal samples were obtained from 120 persons who had visited the diagnostic department of Yonsei University College of Medicine, and they had a mean age of 25 years. Plasma samples were collected over 15 months. Early samples collected from 100 kidney cancer patients and 90 normal persons were used in a preparative experiment (training group sample study) for analyzing the diagnostic performance of the markers. Samples taken later from 28 patients and 30 normal persons were used in a validity test for the diagnostic peformance of the marker obtained in the preparative experiment.
  • a blood sample was collected using a 5 mL sterile vacuum blood collection tube containing 4 mg of K 2 EDTA. After being mixed with the anticoagulant, the blood sample was centrifuged for 10 min at 2,500 rpm to separate plasma as a supernatant, which was then stored at -80°C until experiment.
  • recombinant proteins serving as a calibrator, and antibodies and the biotinylation of a detection antibody were carried out as described previously [ Journal of Proteome Research 2010, 9:3710-3719 ].
  • the recombinant proteins were prepared from full-length cDNAs for NNMT (GenBank accession number: NM_006169), LCP1 (GenBank accession number: NM_002298.4), and NM23A (GenBank accession number: NM_198175.1).
  • Conjugation of an antibody to beads was achieved as follows. First, anti-NNMT IgG was conjugated to bead No. 63; anti-LCP1 IgG to bead No. 17; and anti-NM23A IgG to bead No. 33. Conjugation between an antibody and carboxy-coated beads was made according to the protocol of the manufacturer (Luminex Corp.). Briefly, 1 ⁇ 10 6 beads were washed twice with deionized water by centrifugation, and suspended in 8 ⁇ l of sodium phosphate buffer (pH 6.2) by vortexing and sonication.
  • each single marker assay was evaluated separately and pairwise addition of bead-conjugated capture antibody was conducted. Consistency or changes of a calibration curve in the subsequent addition of a capture antibody was observed. If there was any change in the calibration curve, it was adjusted and optimized by incorporating a set of blocking conditions against nonspecific binding of individual antibody pairs. Twenty microliters of an assay buffer containing 1,000 capture antibody-conjugated beads was transferred, together with 20 ⁇ L of plasma samples or calibrators, to the filterplate. After 30 min of incubation, 10 ⁇ L of biotin-labeled detection antibody (160 ⁇ g/mL) diluted with an assay buffer was added and incubated for an additional 30 minutes.
  • a washing solution 50 mM Tris HCl pH 7.4, 150 mM NaCl, 1 % BSA, 0.1 % sodium azide, 0.05 % Tween 20
  • 50 mL of phycoerythrin-labeled streptavidin (PE,4 mg/mL) in a PE solution was added and incubated for 30 minutes. Without wash, 50 ⁇ L of a PE solution was added and beads were mixed thoroughly before reading on Luminex 100 system (Luminex Corp.). The data were processed and analyzed using using MasterPlex CT software (Ver. 1.0; MiraiBio, Inc.) with linear regression curves.
  • Scoring was carried out as reported previously [ Proc. Natl.Acad. Sci. U.S.A. 2005, 102, 7677-682 ], with the exception for a procedure for best cut-point (criterion) determination of each marker.
  • the best cut-points were selected at the point of maximum AUC of each marker from the ROC analysis.
  • individuals were assigned score 0 ( ⁇ cut-point) or 1 (>cut-point) for each marker and finally assigned a score ranging from 0 to 3 as the sum of 3 markers.
  • Plasma concentrations of the individual markers were measured in a total of 190 plasma samples (90 normal individuals and 100 patients with kidney cancer) and the mean values are depicted in FIG. 1 .
  • the median NNMT concentration in patients with kidney cancer was 420 pg/mL, which is approximately 6.2-fold higher than 68 pg/mL for normal individuals.
  • the median concentration of LCP1 in control individuals and patients with kidney cancers was 10,385 and 13,789 pg/mL, respectively, with an approximately 1.3-fold difference therebetween.
  • NM23A a median concentration of 780 pg/ml and 3,442 pg/ml in control individuals and patients with kidney cancer were measured, respectively, with an approximately 4.4-fold difference therebetween.
  • the sensitivity was 69 % for NNMT, 39 % for LCP1, and 48 % for NM23A, as measured by ROC curve analysis.
  • stepwise logistic regression analysis was conducted to evaluate the diagnostic performance of the combined markers.
  • the predictive probability was generated and used to estimate the diagnostic value by ROC analysis.
  • the diagnostic accuracy (AUC) of logistic regression with the combined markers was 0.933 and the sensitivity at fixed specificity of 95 % was 71.6 %.
  • the diagnostic accuracy of NNMT which exhibited the highest diagnostic performance among the three individual markers, was measured to be 0.927, with a sensitivity of as low as 69 % at a fixed specificity of 95 %.
  • a scoring method was applied in the preparative experiment with a total of 190 samples (90 normal individuals and 100 patients with kidney cancer) to generate predictive diagnostic values.
  • a cut-point (criterion) for each of the three markers was selected at an ROC curve point at a maximum AUC was obtained.
  • the cut-point was determined to be 148 pg/ml for of NNMT, 12974 pg/ml for LCP1, and 1230 pg/ml for NM23A.
  • concentration of each of the three markers was above the cut-point, a score of 1 was assigned; otherwise, a score of 0 was assigned to the subjects, with a finally assigned score ranging from 0 to 3 as the sum of 3 markers.
  • the cut-off value as a criterion for kidney cancer was 1.
  • the diagnostic accuracy of the score was measured to be 0.948 (AUC), with a sensitivity of 89 % at a fixed specificity of 95 %.
  • blinded test group samples including 30 normal samples and 29 patient samples were applied to validate the clinical performance of the three combined markers with the scoring method based on the cut-point determined previously.
  • the combined markers were improved in both diagnostic accuracy (AUC) and sensitivity at 95 % specificity, compared to individual markers.

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EP12884039.4A 2012-09-07 2012-09-27 Utilisation de molécules de liaison spécifiques et d'un kit de détection de biomarqueurs sanguins du cancer du rein Not-in-force EP2894474B1 (fr)

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KR1020120099276 2012-09-07
PCT/KR2012/007846 WO2014038744A1 (fr) 2012-09-07 2012-09-27 Procédé et trousse pour la détection de biomarqueurs sanguins du cancer rénal

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EP2894474A4 EP2894474A4 (fr) 2016-04-13
EP2894474B1 EP2894474B1 (fr) 2018-08-15

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CN105759032A (zh) * 2016-03-18 2016-07-13 南昌大学 一种针对大肠杆菌o157：h7的检测方法

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CN105354445A (zh) * 2015-11-17 2016-02-24 南昌大学第二附属医院 一种基于血液标志物的人工神经网络智能判别系统
CN105759032A (zh) * 2016-03-18 2016-07-13 南昌大学 一种针对大肠杆菌o157：h7的检测方法

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EP2894474B1 (fr) 2018-08-15
WO2014038744A1 (fr) 2014-03-13
CN104718455A (zh) 2015-06-17
CN104718455B (zh) 2017-03-08
EP2894474A4 (fr) 2016-04-13

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